Cancer is a disease resulting from multiple changes at the genomic level. These changes ultimately lead to the malfunction of cell cycle machinery and finally to autonomous cell proliferation. Neoplastic transformation involves four types of genes: oncogenes, tumor-suppressor genes, mutator genes, and apoptotic genes. Different types of cancer can involve alteration of any one or any combination of these genes.
Proto-oncogenes of the myc family are overexpressed in many different types of human tumors including tumors of the breast, colon, cervix, head and neck, and brain. Many solid tumors amplify or overexpress c-myc, with up to a 50-fold increase in c-myc RNA in tumor cells relative to normal cells having been reported (Yamada, H. et al. 1986. Jpn. J. Cancer Res. 77:370-375). For example, three of the six most common solid tumors, including up to 100% of colon adenocarcinomas, 57% of breast cancers, and 35% of cervical cancers, demonstrate increased levels of c-myc protein. Enforced expression of c-myc in nontumorigenic cells causes immortalization but not transformation; however, elevated levels of c-myc protein are rare in benign cancers and normal differentiated tissue. While solid tumors can oftentimes be removed surgically, overexpression of c-myc has been linked with amplification of the c-myc gene and correlated with poor prognosis and an increased risk of relapse (Nagai, M. A. et al. 1992. Dis. Colon Rectum 35:444-451; Orian, J. M. et al. 1992. Br. J. Cancer 66:106-112; Riou, G. et al. 1987. Lancet 2:761-763; Field, J. K. et al. 1989. Oncogene 4:1463-1468).
Another member of the myc oncogene family, N-myc, has been linked with development of neuroblastomas in young children. Overexpression of this member of the myc family of proto-oncogenes has also been correlated with advanced stages of disease and poor prognosis (Brodeur, G. M. et al. 1997. J. Ped. Hematol. Oncol. 19:93-101). Primary tumors for this specific condition usually arise in the abdomen and as many as 70% of patients have bone marrow metastases at diagnosis (Matthay, K. E. 1997. Oncology 11:1857-1875). Treatment of children with Stage 4 disease using surgery, chemotherapy, and purged autologous or allogeneic marrow transplant produces a progression-free survival rate of 25 to 49% in patients four years post transplant (Matthay, K. K. et al. 1994. J. Clin. Oncol. 12:2382-2389). Most relapses after autotransplant occur at sites of bulk disease and/or previously involved sites. Estimates of the rate of local recurrence vary depending upon the study. However, recurrence of tumor at an original site has been estimated to occur in approximately 25% of high risk neuroblastoma patients.
Further, definitive evidence from gene marking studies indicates that autologous marrow, free of malignant cells by standard clinical and morphologic criteria, contributes to relapse at both medullary and extramedullary sites (Rill, D. R. et al. 1994. Blood 84:380-383). In a recent pilot clinical study, bone marrow involvement at diagnosis correlated with specific relapse at that site in children receiving autologous purged marrow (Matthay, K. K. et al. 1993. J. Clin. Oncol. 11:2226-2233). Accordingly, improvements in surgery, detection of tumor margins, development of new anticancer drugs or application of novel therapies are required to prevent local tumor regrowth. In particular, more effective treatment strategies are needed for elimination of “minimal residual disease” or “MRD” which results from the presence of a small number of tumor cells at the site of disease after treatments such as tumor resection or purging bone marrow of tumor cells.
CPT-11 (irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) is a prodrug currently under investigation for the treatment of cancer that is converted to the active drug known as SN-38 (7-ethyl-10-hydroxy-camptothecin) (Tsuji, T. et al. 1991. J. Pharmacobiol. Dynamics 14:341-349; Satoh, T. et al. 1994. Biol. Pharm. Bull. 17:662-664). SN-38 is a potent inhibitor of topoisomerase I (Tanizawa, A. et al. 1994. J. Natl. Cancer Inst. 86:836-842; Kawato, Y. et al. 1991. Cancer Res. 51:4187-4194), an enzyme whose inhibition in cells can result in DNA damage and induction of apoptosis (Hsiang, Y.-H. et al. 1989. Cancer Res. 49:5077-5082). The specific enzyme responsible for activation in vivo of CPT-11 has not been identified, although serum or liver homogenates from several mammalian species have been shown to contain activities that convert CPT-11 to SN-38 (Tsuji, T. et al. 1991. J. Pharmacobiol. Dynamics 14:341-349; Senter, P. D. et al. 1996. Cancer Res. 56:1471-1474; Satoh, T. et al. 1994. Biol. Pharm. Bull. 17:662-664). Uniformly, these activities have characteristics of carboxylesterase (CE) enzymes (Tsuji, T. et al. 1991. J. Pharmacobiol. Dynamics 14:341-349; Senter, P. D. et al. 1996. Cancer Res. 56:1471-1474; Satoh, T. et al. 1994. Biol. Pharm. Bull. 17:662-664). In fact, SN-38 can be detected in the plasma of animals and humans minutes after the administration of CPT-11 (Stewart, C. F. et al. 1997. Cancer Chemother. Pharmacol. 40:259-265; Kaneda, N. et al. 1990. Cancer Res. 50:1715-1720; Rowinsky, E. K. et al. 1994. Cancer Res. 54:427-436), suggesting that a CE enzyme present in either serum or tissues can convert the camptothecin analog to its active metabolite.
CEs are ubiquitous serine esterase enzymes that are thought to be involved in the detoxification of a variety of xenobiotics. CEs are primarily present in liver and serum, however, the physiological role of this class of enzymes has yet to be identified. A recent biochemical analysis of 13 CEs compared their ability to metabolize CPT-11 to SN-38. While the efficiency of conversion varied between enzymes, those isolated from rodents were the most efficient (Satoh, T. et al. 1994. Biol. Pharm. Bull. 17:662-664). The amino acid sequence of a rabbit liver CE has been disclosed (Korza, G. and J. Ozols. 1988. J. Biol. Chem. 263:3486-3495). In addition, there are currently 13 cDNA sequences encoding CE in the GenBank and EMBL databases, including a rat serum and rat liver microsomal CE. Interestingly, CEs purified from human tissues demonstrated the least efficient conversion of CPT-11 to SN-38, with less than 5% of the prodrug being converted to active drug (Leinweber, F. J. 1987. Drug Metab. Rev. 18:379-439; Rivory, L. P. et al. 1997. Clin. Cancer Res. 3:1261-1266).
In addition to metabolism to SN-38, in humans CPT-11 is also metabolized to a compound known as APC (Haaz, M. C. et al. 1998. Cancer Res. 58:468-472). APC has little, if any, anti-tumor activity and is not converted to an active metabolite in humans (Rivory, L. P. et al. 1996. Cancer Res. 56:3689-3694).
In preclinical studies, CPT-11 administered to immune-deprived mice bearing human tumor xenografts produces complete regression of glioblastomas, rhabdomyosarcomas (RMS), neuroblastomas, and colon adenocarcinomas (Houghton, P. J. et al. 1995. Cancer Chemother. Pharmacol. 36:393-403; Houghton, P. J. et al. 1993. Cancer Res. 53:2823-2829). However, maintenance of tumor regression in studies with CPT-11 appears to be dependent upon drug scheduling, suggesting that viable tumor cells survive therapy (i.e., minimal residual disease (MRD)). These studies also showed a steep dose-response relationship between dose of drug administered and induction of tumor regression. For example, 20 mg of CPT-11/kg/day given daily for 5 days for two weeks produced complete regression of Rh18 RMS xenografts, while 10 mg/kg/day given on the same schedule produced only partial tumor regression. Similar effects were seen when mice bearing SJGC3A colon adenocarcinoma xenografts were treated with 40 mg CPT-11/kg compared to a 20 mg/kg dose.
Early clinical trials with CPT-11 indicate that the prodrug also has anti-tumor activity in vivo against many different types of solid tumors in humans. However, myelosuppression and secretory diarrhea limit the amount of drug that can be administered to patients. Accordingly, before this promising anti-cancer agent can be used successfully, these dose-limiting toxicities must be overcome.
The development of new effective treatment strategies for cancer is dependent upon the availability of specific drug screening assays. Specific drug screening assays can involve isolated target tissue models, i.e., isolated heart, ileum, vasculature, or liver from animals such as rabbits, rats, and guinea pigs, wherein the target tissue is removed from the animal and a selected activity of that target tissue is measured both before and after exposure to the candidate drug. An example of a selected activity measured in drug screening assays to identify new cancer agents is the activity of enzymes such as topoisomerase I or II, which are known to modulate cell death. Such assays can also be used to screen for potential prodrugs which are converted to the active metabolite in selected tissues or to identify selected tissues capable of converting prodrug to its active metabolite.
However, any molecular event that is shown to be modified by a novel class of compounds can be developed as a screening assay for selection of the most promising compounds for therapeutic development. In fact, in recent years the idea of modulating cells at the genomic level has been applied to the treatment of diseases such as cancer. Gene therapy for treatment of cancer has been the focus of multiple clinical trials approved by the National Institutes of Health Recombinant DNA Advisory Committee, many of which have demonstrated successful clinical application (Hanania et al. 1995. Am. Jour. Med. 99:537-552; Johnson et al. 1995. J. Am. Acad. Derm. 32(5):689-707; Barnes et al. 1997. Obstetrics and Gynecology 89:145-155; Davis et al. 1996. Current Opinion in Oncology 8:499-508; Roth and Cristiano 1997. J. Natl. Canc. Inst. 89(l):21-39). To specifically target malignant cells and spare normal tissue, cancer gene therapies must combine selective gene delivery with specific gene expression, specific gene product activity, and, possibly, specific drug activation. Significant progress has been made in recent years using both viral (retrovirus, adenovirus, adeno-associated virus) and nonviral (liposomes, gene gun, injection) methods to efficiently deliver DNA to tumor sites. Genes can be transfected into cells by physical means such as scrape loading or ballistic penetration, by chemical means such as coprecipitation of DNA with calcium phosphate or liposomal encapsulation; or by electro-physiological means such as electroporation. The most widely used methods, however, involve transduction of genes by means of recombinant viruses, taking advantage of the relative efficiency of viral infection processes. Current methods of gene therapy involve infection of organisms with replication-deficient recombinant viruses containing the desired gene. The replication-deficient viruses most commonly used include retroviruses, adenoviruses, adeno-associated viruses, lentiviruses and herpes viruses. The efficacy of viral-mediated gene transfer can approach 100%, enabling the potential use of these viruses for the transduction of cells in vivo.
Adenovirus vector systems in particular have several advantages. These include the fact that non-dividing cells can be transduced; transduced DNA does not integrate into host cell DNA, thereby negating insertional mutagenesis; the design of adenoviral vectors allows up to 7 kb of foreign DNA to be incorporated into the viral genome; very high viral titers can be achieved and stored without loss of infectivity; and appropriate plasmids and packaging cell lines are available for the rapid generation of infectious, replication-deficient virus (Yang, N. S. 1992. Crit. Rev. Biotechnol. 12:335-356). The effectiveness of adenoviral-mediated delivery of genes into mammalian cells in culture and in animals has been demonstrated.
To increase the specificity and safety of gene therapy for treatment of cancer, expression of the therapeutic gene within the target tissue must also be tightly controlled. For tumor treatment, targeted gene expression has been analyzed using tissue-specific promoters such as breast, prostate and melanoma specific promoters and disease-specific responsive promoters such as carcinoembryonic antigen, HER-2/neu, Myc-Max response elements, DF3/MUC. Dachs, D. U. et al. 1997. Oncol. Res. 9(6-7):313-25. For example, the utility of herpes simplex virus thymidine kinase (HSV-TK) gene ligated with four repeats of the Myc-Max response element, CACGTG (SEQ ID NO:22), as a gene therapy agent for treatment of lung cancer with ganciclovir was examined in c-, L- or N-myc-overexpressing small cell lung cancer (SCLC) cell lines (Kumagai, T. et al. 1996. Cancer Res. 56(2):354-358). Transduction of the HSV-TK gene ligated to this CACGTG (SEQ ID NO:22) core rendered individual clones of all three SCLC lines more sensitive to ganciclovir than parental cells in vitro, thus suggesting that a CACGTG-driven HSV-TK gene may be useful for the treatment of SCLC overexpressing any type of myc family oncogene. Additional experiments with c-myc have focused on the use of the ornithine decarboxylase (ODC) promoter gene. Within the first intron of the ODC gene are two CACGTG “E boxes” that provide binding sites for the c-myc protein when bound to its partner protein known as max. Mutation of the E box sequence results in the inability of c-myc to transactivate the ODC promoter. Previous reports indicate that reporter constructs containing the ODC promoter fused upstream of the chloramphenicol acetyltransferase gene immediately adjacent to the second exon were activated in cells that overexpress c-myc (Bello-Fernandez, C. et al. 1993. Proc. Natl Acad. Sci. USA 90:7804-7808). In contrast, transient transfection of promoter constructs in which the E boxes were mutated (CACGTG (SEQ ID NO:22) to CACCTG (SEQ ID NO:25) demonstrate significantly lower reporter gene activity. These data suggest that it is possible to activate transcription of specific genes under control of the c-myc responsive ODC promoter. In the case of N-myc, N-myc protein is a basic helix-loop-helix (BHLH) protein that can dimerize with proteins of the same class. N-myc dimerizes with the BHLH protein max to form a complex that binds to the CACGTG motif present in gene promoters, such as ODC, resulting in transactivation and expression of specific genes containing this sequence (Lutz, W. et al. 1996. Oncogene 13:803-812). Studies in a neuroblastoma cell line and tumors have shown that binding of N-myc to its consensus DNA binding sequence correlates with N-myc expression, data that indicate that the level of N-myc in neuroblastoma cells is a determining factor in expression of proteins under control of promoters containing the CACGTG sequence (Raschella, G. et al. 1994. Cancer Res. 54:2251-2255). Inhibition of expression of the c-myc gene via antisense oligonucleotides as a means for inhibiting tumor growth has also been disclosed (Kawasaki, H. et al. 1996. Artif. Organs 20(8):836-48).
In the present invention, polynucleotides encoding carboxylesterase enzymes or active fragments thereof and polypeptides encoded thereby which are capable of metabolizing the chemotherapeutic prodrug CPT-11 and its inactive metabolite APC to active drug SN-38 are disclosed. Use of these enzymes in combination with APC renders this inactive metabolite a useful chemotherapeutic prodrug. It has also been found that compositions comprising a polynucleotide of the present invention and a disease-specific responsive promoter can be delivered to selected tumor cells to sensitize the tumor cells to the chemotherapeutic prodrug CPT-11, thereby inhibiting tumor cell growth.